4-1BBL−/− mice exhibit normal primary CD8 T cell responses to influenza virus, but show decreased CD8 T cell numbers late in the primary response as well as decreased secondary responses. In contrast, CD28−/− mice are defective in initial CD8 T cell expansion. Using agonistic anti-4-1BB Ab to replace the CD28 or 4-1BB signal, we examined the timing of the required signals for CD28 vs 4-1BB costimulation. A single dose of agonistic anti-4-1BB Ab added only during priming restores the secondary CD8 T cell response in CD28−/− mice. Once the T cell numbers in the primary response reach a minimum threshold, a full secondary response is achieved even in the absence of CD28. In contrast, anti-4-1BB added during priming fails to correct the defective secondary response in 4-1BBL−/− mice, whereas addition of anti-4-1BB during challenge fully restores this response. Thus, there is a switch in costimulatory requirement from CD28 to 4-1BB during primary vs recall responses. Adoptive transfer studies show that T cells primed in 4-1BBL−/− or wild-type mice are equally capable of re-expansion when rechallenged in wild-type mice. These studies rule out a model in which signals delivered through 4-1BB during priming program the T cells to give a full recall response and suggest that 4-1BB-4-1BBL interactions take place at later stages in the immune response. The results indicate that anti-4-1BB or 4-1BBL therapy will be most effective during the boost phase of a prime-boost vaccination strategy.

The mechanisms that allow the maintenance of immunological memory remain incompletely defined. Here we report that tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 1, a protein recruited in response to several costimulatory TNFR family members, is required for maximal CD8 T cell responses to influenza virus in mice. Decreased recovery of CD8 T cells in vivo occurred under conditions where cell division was unimpaired. In vitro, TRAF1-deficient, antigen-activated T cells accumulated higher levels of the proapoptotic BH3-only family member Bim, particularly the most toxic isoform, Bim S. In the presence of excess IL-15, memory phenotype T cells with similar surface phenotype and comparable levels of Bcl-2 family members could be generated from WT or TRAF1-deficient T cell receptor transgenic OT-I T cells. However, when the memory CD8 T cells were allowed to compete for survival signals in the absence of antigen in vivo, the TRAF1-deficient T cells showed decreased recovery compared with TRAF1-sufficient T cells. This defect in T cell recovery in vivo was alleviated by introduction of siRNA to down-modulate Bim in TRAF1-deficient memory T cells. These studies identify the TRAF1 signaling axis and Bim down-regulation as critical for CD8 memory T cell survival in vivo.Bcl-2 family ͉ influenza virus ͉ knockout/transgenic mice

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. T he CD8 T cells are important mediators of protective immunity to viruses (1). Indeed a strong CD8 T cell response to influenza offers the hope of cross-protective immunity to diverse influenza strains (2, 3). However, there is a concern that too strong an immune response in the lung will cause immune pathology and a worse outcome (4 -6). Upon initial encounter with Ag, T cells commit to programmed expansion, which occurs independently of the continued presence of Ag (7,8). How then does the immune system provide the appropriate level of T cell stimulation for a particular infection? T cells regulate their level of response by integrating signals from the Ag-specific receptor as well as from costimulatory, coinhibitory, and cytokine receptors which in turn influence the initiation, duration, and differentiation of the response (9 -12). The initial activation of T cells requires costimulatory signals from CD28, expressed on resting T cells. In addition, during an on-going immune response, other costimulatory receptors and ligands are up-regulated on the T cells and APCs (13-15). The question arises as to why so many costimulatory receptor ligand pairs have accumulated in the mammalian immune system. Here we provide evidence that the expression of one such costimulatory receptor, 4-1BB, is sustained in response to severe as compared with mild respiratory infection with influenza virus, thereby allowing the immune system to maintain a level of CD8 T cell response appropriate for the severity of the infection.The receptor 4-1BB (CD137) is a member of the TNFR family expressed on Ag-receptor activated T cells (14, 16 -18). In vivo with nonreplicating immunogens, 4-1BB is rapidly and transiently up-regulated on T cells upon immunization, before the first cell division and concomitantly with the expression of the early activation marker CD69 (19). Evidence that 4-1BB can play a role early in the response comes from systemic administration of agonist anti-4-1BB Abs, which can enhance initial antitumor and antiviral T cell responses wi...

The SLP-76 family of immune cell-specific adaptors is composed of three distinct members named SLP-76, Blnk, and Clnk. They have been implicated in the signaling pathways coupled to immunoreceptors such as the antigen receptors and Fc receptors. Previous studies using gene-targeted mice and deficient cell lines showed that SLP-76 plays a central role in T-cell development and activation. Moreover, it is essential for normal mast cell and platelet activation. In contrast, Blnk is necessary for B-cell development and activation. While the precise function of Clnk is not known, it was reported that Clnk is selectively expressed in mast cells, natural killer (NK) cells, and previously activated T-cells. Moreover, ectopic expression of Clnk was shown to rescue T-cell receptor-mediated signal transduction in an SLP-76-deficient T-cell line, suggesting that, like its relatives, Clnk is involved in the positive regulation of immunoreceptor signaling. Stimulatory effects of Clnk on immunoreceptor signaling were also reported to occur in transfected B-cell and basophil leukemia cell lines. Herein, we attempted to address the physiological role of Clnk in immune cells by the generation of Clnkdeficient mice. The results of our studies demonstrated that Clnk is dispensable for normal differentiation and function of T cells, mast cells, and NK cells. Hence, unlike its relatives, Clnk is not essential for normal immune functions.The SLP-76 family of adaptors includes three related polypeptides named SLP-76, Blnk (also termed SLP-65 or BASH), and Clnk (also named MIST) (14,18,28). These intracytoplasmic molecules share a common organization, which includes an amino-terminal acidic domain carrying sites of tyrosine phosphorylation, a central region with proline-rich motifs and arginine-rich sequences implicated in protein-protein interactions, and a carboxyl-terminal Src homology 2 (SH2) domain. Accumulating evidence indicates that SLP-76 and Blnk orchestrate the formation of multimolecular complexes leading to activation of phospholipase C␥, as well as of the guanine nucleotide exchange factors Vav-1 and Sos, in response to stimulation of immunoreceptors including the antigen receptors and Fc receptors.